Tissue engineered blood vessels and apparatus for their manufacture

ABSTRACT

The invention is a tissue engineered blood vessel (TEBV) made from a cultured fibroblast sheet rolled into a multilayer vessel which has sufficient burst strength to withstand physiological blood pressure without the inclusion of smooth muscle cells or synthetic scaffolding. The TEBV is made in a bioreactor having an enclosed chamber, a sheet growth module, a rollable mandrel, and a clamp for holding the sheet to the mandrel for rolling.

CROSS-REFERENCE

This application is a divisional application of and claims priority toU.S. application Ser. No. 09/444,520, filed on Nov. 22, 1999, now U.S.Pat. No. 6,503,273 the entire disclosure of which is incorporated byreference.

BACKGROUND AND PRIOR ART

This invention relates to a method and apparatus for the fabrication ofautologous blood vessels without smooth muscle cells or non-biologicalscaffolding materials. The principal use of such blood vessels is forsmall diameter bypass grafts such as coronary artery bypass graft(CABGs), peripheral bypass grafts, or arteriovenous shunts.

Despite dramatic declines in the coronary heart disease related deathsover the last 30 years, heart disease remains the number one cause ofdeath in the industrialized world. The decline in the death rate hascoincided with the development and success of CABGs. In this procedure,vessels are typically harvested from the patient's own internal mammaryarteries or saphenous veins (called an “autologous graft”) and graftedinto the coronary circulation to bypass arteries occluded byatherosclerotic plaques.

One important limitation of this procedure is that grafted vessels aresusceptible to plaque regrowth leading to blockages (restenosis). MostCABGs using internal mammary arteries remain functional for 10–12 years.Beyond this timeframe, restenosis rates increase dramatically. Manypatients therefore require a second bypass. In 1995, 573,000 grafts wereperformed in the U.S. alone, and 43% of these were in patients between45 and 64 years of age, suggesting that a large fraction of theseyounger patients will live long enough to require a second procedure.However, due to previous vessel harvest or to vascular diseaseprogression, suitable vessels for further arterial reconstruction oftenare not available. Currently, there is no clinically viable long-termtreatment strategy for these patients. Over 50,000 Americans dieannually due to the lack of graftable vessels. This number is likely tocontinue to rise, lagging 10 years behind the exponential growth inprimary CABG procedures.

Prior attempts at finding alternate vessel sources focused on syntheticmaterials or dried and fixed tissue transplants. Larger diametersynthetic grafts have demonstrated reasonable functionality in theperipheral circulation. In the small diameter (less than 6 mm insidediameter) coronary circulation, however, synthetic grafts do not workand typically fail due to thrombogenesis (the formation of blood clotsthat occlude the vessel). Moreover, the synthetic materials ofteninitiate chronic inflammatory responses that may be the cause of vesselfailure.

In an effort to improve synthetic graft integration, syntheticbiomaterials have been combined with autologous tissues from thepatient. One procedure employs a tubular silicone mandrel surrounded bya Dacron mesh. This was implanted beneath the skin in patients scheduledfor vascular reconstruction. After two months, the implant was removedfrom the patient and the Dacron mesh, along with a tightly integratedfibrous tissue, was slid away from the mandrel. This Dacron-supportedscar tissue was then utilized as a vascular graft in the same patient.Although this graft combined autologous living cells and a naturalextracellular matrix with a synthetic Dacron support, it still faileddue to clotting.

Cell-seeded conduits have been tried that require resorbable,non-biological scaffolds to generate sufficient mechanical strength. Inthis approach, cells (typically smooth muscle cells) are seeded intotubular structures made from materials such as polylactic acid. Thesevessels are susceptible to the same thrombosis/inflammation failuresassociated with an immune response. It is also difficult to exactlymatch their biomaterial degradation rates with tissue remodeling.Vascular grafts made from synthetic materials also are prone tobacterial colonization and infection, which can result in loss offunction or serious systemic complications.

Furthermore, these vessels provide no mechanism to limit theproliferation of smooth muscle cells. Smooth muscle proliferation mayinfiltrate the lumen of the vessel and occlude it in a process calledintimal hyperplasia. Finally, synthetic grafts have very differentmechanical properties from natural tissues. These differences,particularly in tissue compliance, may induce adverse remodelingresponses and graft failure due to localized non-physiologicalhemodynamic forces.

One promising solution to the problems associated with synthetic-basedvascular grafts is to assemble blood vessels in vitro using only thepatient's own cells and then re-implant them into the patient. Thisapproach is called tissue engineering. In theory, tissue-engineeredblood vessels (TEBVs) should provide mechanically stable vessels builtonly from autologous tissue, therefore generating no immune responses.Another advantage to a tissue engineering approach is the ability tomanipulate the vessel ends to facilitate grafting of the vessel intoplace. Tissue engineering has been used successfully in the past tobuild two-dimensional structures such as skin, but has had only limitedsuccess with three dimensional tissues and organs such as TEBVs. Themost common problem with three-dimensional engineered tissues is a lackof structural integrity and mechanical strength. This is a particularproblem for TEBVs, since these vessels will be subjected to significantmechanical loads both from blood pressure (which may be abnormally highin patients with heart disease), as well as relative motion between theanchoring sites of the vessel (the aorta and the myocardium). Moreover,the TEBVs must demonstrate sufficient suturability and tear-resistanceto allow surgical handling and implantation.

Recently, a tissue engineering technique has been developed that is aradical departure from prior art techniques. This work is described inU.S. Pat. No. 5,618,718 and in an article entitled “A CompletelyBiological Tissue-Engineered Human Blood Vessel,” L'Heureux, N., et al.,FASEB J. 12:47–56, 1998, both of which are incorporated herein byreference. A fully biological and autologous human TEBV, with nosynthetic materials, was made and found capable of withstandingphysiological burst pressures in excess of 2000 mm Hg. These vesselswere suturable and maintained patency for one week when xenografted intoa dog. A living graft of this type is self-renewing with an inherenthealing potential. The completely biological graft can be remodeled bythe body according to the demands of the local mechanical environment.Moreover, the absence of synthetic materials precludes foreign bodyreactions, thus increasing the likelihood of long-term graft success.

These prior art TEBVs are prepared by rolling sheets of culturedfibroblasts and smooth muscle cells around a tubular mandrel. After a2–8 week maturation period, the mandrel is removed and the vessel isseeded with endothelial cells. In this prior art procedure, the smoothmuscle cells provide the TEBV with the ability to emulate a real bloodvessel's ability to expand and contract. The smooth muscle cells alsoare responsible for the superior burst strength which in the past hadbeen provided by the synthetic materials or scaffolds.

Even these prior art fully autologous TEBVs have significant problems.One of the biggest problems with TEBVs made from smooth muscle tissue isthe difficulty associated with detaching and rolling the sheets oftissue while maintaining uniform vessel thickness. The inclusion of asmooth muscle layer complicates and lengthens the overall fabricationtime by at least three weeks. Moreover, smooth muscle cells tend touncontrollably proliferate. This undesired proliferation of cells mayocclude the vessel. Moreover, the prior art rolling and tissuefabrication processes need to be improved and automated in order toobtain a clinically viable TEBV required for bypass grafts.

Contrary to the expectations in the art, a mechanically stable and fullyautologous TEBV structure was discovered which could be fabricatedwithout using smooth muscle tissue. The TEBV of this invention is madefrom fibroblasts and endothelial cells alone, thus eliminating theabove-described problems caused by smooth muscle tissue. The new TEBVstructure and method for its manufacture using an automated bioreactorare described below.

SUMMARY

This invention relates to a tissue engineered blood vessel (TEBV) madefrom a cultured fibroblast sheet rolled into a multilayer vessel whichhas sufficient burst strength to withstand physiological blood pressurewithout the inclusion of smooth muscle cells or synthetic scaffolding.The TEBV of the invention is made in the bioreactor of the inventionhaving an enclosed chamber, a sheet growth module, a rollable mandreland a clamp, preferably a magnetic clamp, for holding the sheet to themandrel during rolling and maturation.

The process for making the TEBV of the invention begins with isolatingand expanding fibroblasts from an autologous biopsy in a culture,producing a sheet of cells and extracellular matrix. The cell sheet isrolled onto a mandrel to produce a rolled vessel, which is then maturedto make a TEBV.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a perspective view of the bioreactor of the invention, showingthe chamber enclosure as transparent;

FIG. 2 a is a side view of the detaching mandrel of the invention;

FIG. 2 b is a cross-section of the detaching mandrel of FIG. 2 a;

FIGS. 3 a, 3 b, 3 c, and 3 d are cross-sectional views which show thetissue rolling process of the invention; and

FIGS. 4 a and 4 b are perspective and cross-sectional views,respectively, of the TEBV of the invention rolled on a mandrel.

DETAILED DESCRIPTION

The first step in the process of making the TEBV of this invention is toprepare a sheet of fibroblasts. This sheet includes fibroblasts takenfrom the patient's own body (an autologous graft) or from donor tissue(an allograft). Allograft tissues have the advantage of being able totreat many patients from a single donor source. However the cells usedin allograft tissue must be treated to eliminate physiological foreignbody responses. This treatment typically includes removing surfaceantigens such that host antibodies cannot label the cells fordestruction. This can be accomplished chemically or enzymatically.

In a preferred embodiment of the invention, autologous cells areharvested from the patient's own body to eliminate the risks of diseasetransmission and tissue rejection. Nearly all tissue biopsies containsome endothelial cells and some fibroblasts. Therefore, almost anybiopsy procedure or tissue harvest will provide a suitable startingpoint for both. Skin or blood vessels are the best tissue sources.

The skin biopsy is accomplished, as is well known in the art, byharvesting from a patient a small patch of skin and fat, and thenisolating the endothelial cells from the capillaries and the fibroblastsfrom the dermis. Blood vessel biopsies can be taken by removing a smallsegment of a peripheral vein or artery, preferably a jugular vein (orsimilar superficial vessel). Alternatively, a small segment can beharvested endoscopically (via a catheter) or by dissecting out a deepvessel. Mesothelial cells harvested from fat can be used in place ofendothelial cells.

Once a suitable biopsy is taken, fibroblasts must be isolated andexpanded to obtain purified cultures. However, reasonably low levels ofother cell types may exist in the purified cultures. Fibroblasts can beisolated from the biopsy by several different well-known techniques. Theeasiest is manual dissection of skin or blood vessels to separate thefibroblast-containing tissue. For skin biopsies, the dermis must beisolated, taking care to remove hair follicles which are a source ofkeratinocyte contamination. For blood vessel biopsies, the adventitiamust be isolated from the media and endothelial layers. Fibroblasts canbe harvested from this portion of the tissue explant by cell outgrowthor by enzymatically digesting the explant and plating the digestedtissue.

Fibroblasts can also be isolated by varying the culture conditions tofavor their growth. Surface material choice (glass versus plastic) orsurface preparation (gelatin or fibronectin coating) can be selected tofavor fibroblast proliferation. Likewise, media additives and pH can beadjusted to promote a preference for endothelial cell or fibroblastproliferation. After a few passages, the cell population will besufficiently pure. Fibroblasts may also be isolated by flow cytometry.In practice, fibroblasts are more difficult to sort by this technique,as there are no clear antibodies that are unique to fibroblasts.

After the fibroblasts have been isolated, they must be cultured andgrown into sheets with sufficient mechanical strength to be detached androlled into three-dimensional conduits. Mature sheets of fibroblasts andtheir extracellular matrix proteins typically take approximately fourweeks to produce, but this time depends upon initial seeding density,media characteristics, and surface preparation. There is somevariability between patients as well. Additional cells can be added tothe fibroblasts at any stage of sheet formation. These cells can includeadditional human or animal cells or transfected or otherwise geneticallymodified cells.

Referring to FIG. 1, sheet growth, detachment, and rolling to make theTEBV are accomplished in bioreactor 40 of this invention. Thisbioreactor is designed specifically for fabrication of TEBVs. Prior artcell culture bioreactors are for the most part designed for cellsuspensions of non-adherent cells or for expansion of cell monolayers.Therefore they do not provide the means of detaching and rollingadherent sheets of tissue required for the TEBVs of this invention.

Sheet growth is carried out in sheet growth module 1 located withinchamber 25 of bioreactor 40. Module 1 may have a lid (not shown).Bioreactor 40 includes automated motion control devices 2 and 3 tofacilitate sheet detachment and rolling, and lid removal, respectively.During sheet formation, non-fibroblast cellular contaminants areminimized, and cells are stimulated to produce a robust extracellularmatrix (ECM). Inducing the formation of an ECM is a critical componentof a fully biological TEBV. There are several ways to stimulateproduction of ECM proteins. In the preferred embodiment, mediaconstituents, such as ascorbic acid, are added to stimulate ECMproduction. Substrate 26 of module 1 preferably has a sterilepolystyrene surface that has been electrostatically treated and coatedwith a protein, such as gelatin or fibronectin, to promote celladhesion. Other surfaces for substrate 26, including glass, acrylic ormetal such as titanium, can be used instead of polystyrene.

Media and gas exchange in chamber 25 is controlled automatically.Chamber 25 includes a one-way valve 8 designed to preserve the sterilitywithin chamber 25 and a drain 7 to remove fluids from chamber 25. Mediaand gas can be metered into and out of chamber 25 through valve 8. Thesheets 27 are maintained in a constant environment throughout the cellexpansion phase. Fibroblasts are seeded into the sheet growth process inthe chamber 25 at an initial density of approximately 10,000 cells percm², although seeding density is not critical to the invention.

After seeding, the first maturation and proliferation phase of the sheetgrowth begins. Fibroblasts will proliferate in almost anyserum-containing cell culture media. A preferred media to optimize thefibroblast proliferation rate and the production of extracellular matrixproteins (which provide mechanical strength to the sheet) is DMEM andHams F12 in a 1:1 ratio, supplemented with 10% fetal calf serum andantibiotics. A preferred embodiment also includes ascorbic acid or otherascorbate derivatives because they accelerate the production of theextracellular matrix proteins.

There are several growth conditions that must be maintained during sheetgrowth in module 1. First, the media pH should be maintained in a rangebetween about 5 and 9, preferably approximately 7.4. Second, the mediatemperature should be maintained in a range between about 25° C. and 45°C., preferably approximately 37° C. Third, a sterile air environmentshould be employed, preferably including up to 20% CO₂. In addition, anadequate media exchange rate must be maintained to prevent exhaustingcritical media constituents.

Bioreactor 40 can be expanded to allow multiple sheets simultaneously tobe fed from one reservoir using multiple modules 1 (only one is shown).Each patient's cells will mature in a separate module fed by a commonmedia source through valve 8. The modules may be isolated from eachother and from the common reservoir by sterile filters andinterconnected passages controlled by solenoid valves (none of which isshown). Alternatively, module 1 can be removed from bioreactor 40 andstored in a separate incubator (not shown). Module 1 includes one-wayvalves (not shown) to exchange media and gases from a common reservoir.The rate of media exchange can be varied between continual and weeklyexchange, but typically between about 25%–100% of the media is exchangedeach day, preferably about 50% being changed every two days.

Referring to FIGS. 1 and 2, sheet growth and maturation module 1 of thisinvention is adapted for the automated rolling steps using an automatedrolling device to roll the TEBVs. Sheets 27 remain in module 1, which isdesigned to be inserted into the bioreactor from port 29 at the front,as shown. The lid (not shown) and one side 28 of module 1 are removableto allow the rolling mandrel 4 and control rod 5 to rest on sheet 27.Alternatively, side 28 may have apertures for the mandrel and rod. Asshown in FIGS. 2 a and 2 b, a preferred embodiment of control rod 5consists of a bladed mandrel with a foam backing.

The rods used for each step are inserted into motion control device 2from the left side. A cartridge 6 of rolling mandrels and/or controlrods can be accessed without human intervention by the motion controldevice 2. When a patient's sheet expansion module is inserted into thebioreactor 40, a sterile set of control rods and/or mandrels 6 is hungfrom the top of the chamber 25, as shown. The motion control device 2can move in three dimensions to access a new mandrel or control rod fromcartridge 6. Control rod 5 preferably has a slot 17 for attachment ofvarious tools. Other options to access and roll the sheet may beemployed that may necessitate additional gearing between the motioncontrol device and the mandrel.

Referring to FIGS. 1, 2 a, 2 b, and 3 a–3 d, the first tool used toinitially separate leading edge 32 of the tissue layer to be rolled fromthe underlying cell substrate is control rod 5 with rubber blade 19attached to it along its length, as shown in cross-section in FIG. 2 b.Blade 19 is rotated against the tissue sheet 27 in a manner to separateabout 1–3 cm of the leading edge 32 of tissue as shown in FIG. 3 b.After separation, leading edge 32 will float slightly and be capable ofbeing lifted and draped over mandrel 4, as shown in FIG. 3 c. Thepreferred mandrel 4 has a surface of Teflon or other material which doesnot allow significant cell adhesion. Alternatively, the surface ofmandrel 4 can be a biological material, biodegradable synthetic compoundor biologically active compound.

Referring to FIGS. 2 a, 2 b, and 3 a–3 d, the next step is to beginrolling sheet 27 around mandrel 4. To facilitate this rolling, a secondtool, for example, sponge 18 mounted on control rod 5 as shown in FIG. 2b, is rotated in contact with the separated edge 32 of tissue sheet 27as shown in FIG. 3 b. This rotation transfers the edge 32 of the tissueto mandrel 4 about which the tissue will be rolled. Once the tissue hasbeen draped over the mandrel 4, as shown in FIG. 3 c, it can be clampedonto mandrel 4 using clamp 15 as shown in FIG. 3 d to facilitate therolling process.

There are a variety of clamping techniques that may be used. A preferredembodiment shown in FIGS. 4 a and 4 b employs a metal clamp 15. Briefly,in accordance with this invention, a magnetic core 14 is inserted intothe inside of mandrel 4 so that the metallic clamp 15 (shown in FIG. 3d) can be held onto mandrel 4 by the field of magnetic core 14. Thisclamping force can be varied by altering the size of the magnet which isused for core 14, the number of magnets used, the strength of themagnets, the separation distance between the core 14 and clamp 15, theferrous content of clamp 15 or the material used for mandrel 4. In apreferred embodiment, the magnetic strength of core 14 can be variedexternally, using methods well known in the art. For example, a magneticfield may be induced in an iron-containing core by applying a currentthrough a coil (not shown) wrapped around core 14. By changing themagnitude of the applied current, the magnetic field can be increased ordecreased proportionally.

Other clamping devices can be utilized for clamp 15, such as amechanical clamp, biological adhesives (fibrin glue) or a slot in themandrel itself, as will be described below. Mechanical clamps can beadded to the edges or across the long axis of mandrel 4 that will usespring force or other type of fastening to secure the rolled tissue 1 tothe mandrel.

Different protein coatings can also be utilized to increase the frictionbetween the mandrel and tissue. This “glue” can later be solubolized ordenatured chemically, enzymatically, acoustically, or thermodynamically.Where the mandrel has a longitudinal slot 17 shown in FIG. 2 a, theleading edge 32 (FIG. 3 b) of the tissue can be dropped into slot 17.The tissue is then held in place by the spring force of mandrel 4, or byinserting the leading edge 32 into a core (not shown) that secures thesheet to the inner surface of mandrel 4.

Mandrel 4 with sheet 27 firmly attached by clamp 15, as shown in FIG. 3d, is then rotated and translated to wind the tissue 16 around it asshown in FIGS. 4 a and 4 b. Motion control device 2 is used to maintainuniform tension and tissue thickness during rolling. The elimination ofbubbles and tension inconsistencies contributes to increased strength ofthe TEBV.

The ends of the mandrel 4 do not have to be cylindrical. They may beflared or tapered to facilitate grafting. Staples or sutures may also beplaced into the TEBV ends to further simplify surgical handling of theTEBVs.

The TEBV can be made with or without an internal membrane. In thepreferred embodiment, an internal membrane is used, made of a sheet offibroblasts that has been air dried to generate dead fibroblasts orotherwise kill the fibroblasts or denatured (decellularized). Afterrolling has been completed, the internal membrane and outer fibroblastplies can be converted from a single sheet of fibroblast by selectivelykilling or denaturing the inner layers by several well-known techniques,including thermal shock, ultrasound, preventing or limiting media or gasexchange on the inner surface, or releasing a localized toxin at theinner surface.

After rolling, clamp 15, which remains inside the roll, can be left inplace or removed. Another clamp 41, shown in FIG. 4 a, may be placedaround the rolled vessel, using any one of the means described above toprevent unwinding.

Next the rolled vessel is transferred to a second module for thematuration phase. This module frees up chamber 25 for rolling otherTEBVs. This maturation phase will last about eight weeks, althoughshorter or longer periods are possible. The sequentially applied layersof the TEBV fuse together during this phase, forming a homogenous TEBV.

There are at least two possible types of maturation reactors. Thesimplest is a passive reactor similar to module 1, which exchanges mediain the manner described in the sheet formation phase. The disadvantageof such a reactor is that vessel thickness is limited by diffusion.Thicker tissues and faster maturation can be generated by using a moduleof a preferred embodiment of the invention which applies a pressuredifferential across the vessel wall. Negative pressure in the mandrelcore is preferred. Placing small perforations in the mandrel, or using aporous material for the mandrel will keep the vessel collapsed againstthe mandrel support. Positive pressure can also be used. Applying apressure gradient across the vessel wall not only increases masstransport, but also will enhance interstitial fluid flow, therebystimulating the cells and increasing the mechanical strength of theTEBV.

When the maturation phase is complete, the mandrel 4 is removed and thevessels are cannulated to introduce endothelial cells. These cells areisolated in a manner similar to fibroblasts. First, the endothelium isseparated from a harvested blood vessel using mechanical force (gentlyscraping the lining of the vessel) or, in a preferred embodiment, usingenzymatic digestion. Collagenase, for example, will preferentiallyrelease the endothelium. Vessels are exposed to an enzyme (collagenase,for example) such that the endothelial layer is preferentially released.

Isolating endothelial cells from skin samples or other tissue samples ismore difficult than isolating fibroblasts, but can be done bycontrolling the culture conditions to favor endothelial cell attachmentand proliferation or by immunological techniques, as is known in theart. Immunological techniques require cell-specific antibodies such thatthe cells of interest (or everything but the cells of interest) arelabeled. Antibody-tagged cells can then be sorted by flow cytometry. Inthis technique, a fluorescent marker is coupled to the antibody so thatcells marked by that antibody fluoresce and can be sorted fromnon-fluorescing cells.

After sufficiently pure cultures of endothelial cells are obtained, thecells must be expanded to provide enough cells to seed the lumen of theTEBV. Endothelial cells may lose important phenotypic characteristicsafter multiple passages. It is therefore important to maximize theinitial harvest such that in vitro expansion is minimized.

After a sufficient quantity of endothelial cells has been obtained, thecells are detached (typically by enzymatic or mechanical means) andplaced into a suspension. The suspension is introduced into the lumen ofthe TEBV via a cannula. After seeding with endothelial cells, thecannula is removed and the TEBV is left in the maturation chamber toallow the endothelium to grow to confluence and anchor securely to thebiological substrate. The resultant TEBVs can be grafted in the patientat this point or, if desired, the inner lining of the vessels may befurther conditioned using hemodynamic forces by introducing fluid flowdown the cannula and through the vessel.

It is preferable to use the TEBVs soon after the endothelial layerreaches confluence because it is difficult to maintain endothelial cellsin a serum-containing culture for long periods of time. However, in somecases, the vessels may be removed from the maturation module andpreserved by freezing, freeze-drying, or preserved by other means.

As will be understood by those skilled in the art, many changes in theapparatus described above may be made by the skilled practitionerwithout departing from the spirit and scope of the invention, whichshould be limited only as set forth in the claims which follow.

1. A TEBV consisting of: a) an inner layer of endothelial cells; b) amiddle layer of denatured fibroblasts; and c) an outer layer of livingfibroblasts.
 2. The TEBV of claim 1 which is prepared as follows: a)providing a first fibroblast layer which is a cultured fibroblast sheetrolled into a multilayer vessel which has been matured and thendenatured; b) rolling a cultured fibroblast sheet around the firstfibroblast layer to form a second fibroblast layer; c) maturing thesecond fibroblast layer; and d) forming a layer of endothelial cells onthe inner surface of the first fibroblast layer.
 3. The TEBV of claim 1wherein the fibroblasts are stimulated to produce an extracellularmatrix (ECM).
 4. The TEBV of claim 1 wherein the fibroblasts areautologous.
 5. The TEBV of claim 1 wherein the fibroblasts areallogenic.
 6. The TEBV of claim 1 wherein the fibroblasts aregenetically modified.
 7. The TEBV of claim 1 wherein the endothelialcells are autologous.
 8. The TEBV of claim 1 having two ends adapted tobe attached to a human blood vessel.
 9. The TEBV of claim 8 whereinstaples are attached to one end.
 10. The TEBV of claim 8 wherein suturesare attached to one end.
 11. The TEBV of claim 8 wherein sutures areattached to both ends of the TEBV.
 12. The TEBV of claim 8 whereinstaples are attached to both ends of the TEBV.
 13. The TEBV of claim 8wherein the ends are tapered.
 14. The TEBV of claim 4, wherein theautologous fibroblasts are obtained from a subject having a vasculardisease.
 15. The TEBV of claim 11 wherein the two ends are flared. 16.The TEBV of claim 1 wherein the fibroblasts are obtained from skin or ablood vessel.
 17. The TEBV of claim 1 wherein the endothelial cells areobtained from skin or a blood vessel.
 18. The TEBV of claim 1 wherein atleast one fibroblast layer is matured after rolling.
 19. The TEBV ofclaim 1 which is prepared by rolling a cultured fibroblast sheet into amultilayer vessel, selectively denaturing at least one inner layer ofthe vessel and forming a layer of endothelial cells on the inner surfaceof the denatured fibroblast layer.
 20. The TEBV of 19 wherein the rolledfibroblast sheet is matured prior to denaturation.